Cancer Nano (2013) 4:13–20 DOI 10.1007/s12645-013-0033-8
ORIGINAL PAPER
Size-dependent cellular toxicity and uptake of commercial colloidal gold nanoparticles in DU-145 cells Pallavi Vedantam & George Huang & T. R. Jeremy Tzeng
Received: 24 January 2013 / Accepted: 20 February 2013 / Published online: 9 March 2013 # Springer-Verlag Wien 2013
Abstract Urinary tract infection (UTI) is a predominant condition in prostate cancer patients. Escherichia coli ORN178 (EC-178) is the uropathogen that causes recurrent infection by binding specifically to adhesins of prostate cancer cells (DU-145 cells). Gold nanoparticles (GNPs) have been used in biodiagnosis of pathogens. In this study, we have investigated the binding time of EC-178 to DU-145 cells, the cytotoxicity and uptake of plain and mannose functionalized and 20 and 200 nm GNPs (D-mannan (Mn)GNPs). We also investigated the protein corona of GNPs when incubated with fetal bovine serum to study the protein corona which decides the biological fate of the GNPs. It was seen that EC-178 binds and is inside the DU-145 cells by 3 h of incubation period. Plain 20 nm GNPs decrease the percentage of viable cells in 48 and 72 h in log and lag phase of DU-145 cells. It was also observed that the Mn-GNPs were taken up by the DU-145 cells significantly more than the plain GNPs. Protein corona was observed when GNPs were incubated with fetal bovine serum which was confirmed by dynamic light scattering measurements and SDS-PAGE gel. Keywords Gold nanoparticles . E. coli . DU-145 cells . Mannose . Urinary tract infection . Cytotoxicity . Biodiagnostic . Protein corona . Cell uptake
1 Introduction The estimated risk of prostate cancer is 21 % and the lifetime risk of death is 2–5 %. Even though it can be diagnosed early and therapy can be started immediately, P. Vedantam (*) : G. Huang : T. R. J. Tzeng Department of Biological Sciences, Clemson University, Clemson, SC 29634, USA e-mail: [email protected]
patients developing metastatic conditions die. Apart from the potent issue of cancer, recurrent urinary tract infection (UTI) is one of the most prevalent symptoms (Klein and Thompson 2012). Escherichia coli ORN178 are the most common cause of UTI in humans. Various urovirulence factors of E. coli ORN178 have been identified such as molecular biology of surface receptors of the urothelial cells of the urinary tract, the adhesin specificity and primarily and the type 1 fimbriae associated with the organism (Sokurenko et al. 1997). Detection, diagnosis, and treatment of UTI play an important role in prostate cancer. One of the novel tools currently vastly studied are nanoparticles. Nanoparticles continue to be used as carriers for localized drug diffusion to treat and detect infections and diseases like cancer (Chen et al. 2008). Owing to their nano size, it is easy for these particles to diffuse into the cells and effect desired responses in treatment of diseases. However, the size, type, and surface charges of the particles play a vital role. Of the different kind of nanoparticles, gold nanoparticles (GNPs) have been extensively studied in this regard. It has been shown that E. coli ORN178 binds specifically to D-mannose, which is an integral part of the glycoproteins that are a part of the adhesive domain on host cells (Sharon 2006). The fimbriae of the uropathogenic E. coli ORN178 bind to the urolapkins on the surface of urothelial cells of the human bladder. We have demonstrated D-mannose functionalized 200 nm GNPs bind specifically to E. coli ORN178 (Vedantam et al. 2012). Continuing in the same direction in this study, we attempt to see if GNPs can be used as specific biodiagnostic tool to detect and treat UTI in prostate cancer cells (DU-145). This study investigates the binding time of E. coli ORN178 and E. coli ORN208 to DU-145 cells. It has been shown that E. coli ORN178 binds specifically to D-mannose only and E. coli ORN208 serves as a negative control as it has type 1 pili that fail to bind to D-mannose. In order to study the cytotoxicity of plain and functionalized 20 and 200 nm GNPs to
14
P. Vedantam et al.
DU-145, growth curve of the prostate cancer cells was done. Cytotoxicity tests in log and lag phase were performed to study their biocompatibility in vitro. Cellular uptake of GNPs was estimated and protein corona of GNPs was studied (Table 1).
Soy Broth with ampicillin (50 μg/ml). The cultures were washed and resuspended in sterile PBS. A 100 μl (3×108 cells/ml) aliquot of EC-178 and 208 was added to two wells each in the chamber slide. The slide was incubated for 1, 2, and 3 h. At the three different time periods, wells were washed with PBS and images were taken by a fluorescent scope (Zeiss LSM-510).
2 Materials and methods 2.2 DU-145 cell growth curve The strains E. coli ORN178 and 208 were provided by Dr. Chu-Cheng Lin, Department of Zoology, National Taiwan Normal University and were transformed with plasmid pGREEN by electroporation (Sambrook and Russell 2001). Two different GNPs: 20 and 200 nm were purchased from Ted Pella Inc., USA. The concentration of 20 nm GNPs was 7 × 1011 particles/ml and 200 nm GNPs was 7× 108 particles/ml. The sugar D-mannan (Mn) was purchased from VWR (USA). The sugar was dissolved in 0.3 M sodium phosphate buffer. Surface functionalization of GNPs with the Mn was carried out by a modified multistep procedure (Aslan et al. 2004; Vedantam et al. 2012). All chemicals required for functionalizing GNPs were purchased from VWR, USA. Human prostate carcinoma cell line DU-145 was graciously given by Dr Arun Sreekumar, Baylor College of Medicine, Houston, TX, USA. Dulbecco’s modified Eagle’s medium (DMEM) was modified to contain Earles Balanced Salt Solution, non-essential amino acids, 2 mM L-glutamine, 1 mM sodium pyruvate, and 1,500 mg/L sodium bicarbonate. It was supplemented with fetal bovine serum to a final concentration of 10 %, 100 UI/ml penicillin G, and 100 μg/ml streptomycin in a humidified incubator with 5 % at 37 °C. All the media components were purchased from Promega, USA. 2.1 Binding of E. coli to DU-145 cells An eight-well chamber slide was used to perform the bacterial cell adhesion assay to DU-145 cells. A total of 0.5 ml of DU-145 cells (1.5×106cells/ml) was seeded in each well. The chamber slide was incubated at 37 °C overnight for attachment and fresh media was added. Fresh cultures of E. coli ORN178 (EC-178) and E. coli ORN208 (EC-208) were cultured overnight in Tryptic Table 1 Hydrodynamic size distribution of GNPs Sample
Size (nm)
Zeta potential (mV)
20 nm GNP Protein coated 20 nm GNP 200 nm GNP Protein coated 200 nm GNP
24±2.1 66.61±2.3 213.5±2.9 261.9±5.3
−54.4 −18.2 −49.3 −13.3
Cells were plated 96-well microtiter plates at initial densities of 1,000, 2,000, 4,000, and 8,000 cells per well. The cell culture medium was changed every 3 days. Cell growth was tested by the CellTiter 96® AQueous One Solution Cell Proliferation Assay (MTS) purchased from Promega, USA. It is a colorimetric method for determining the percentage of viable cells that are proliferating. Briefly, the MTS tetrazolium is bioreduced by the viable cells into a colored formazan product which is stable and can be measured at an absorbance of 490 nm. The amount of formazan produced is directly proportional to the number of living cells in the well. MTS assays were performed every day after seeding until day8. All the experiments were carried out in triplicates. 2.3 Cytotoxicity of GNPs to DU-145 cells The cytotoxicity of plain GNPs was tested by CellTiter 96® AQueous One Solution Cell Proliferation Assay (MTS) purchased from Promega, USA. Cytotoxicity was measured in both the logarithmic and stationary phase of cell growth. For cytotoxicity measurements in the logarithmic phase, each cell line was incubated for 72 h in 96-well plate before adding the GNPs. Fresh medium containing increasing three different concentrations of the GNPs (10, 50, and 100 μl) was added to each well and the cells were incubated for 6, 24, 48, and 72 h time periods. The same was done for testing cytotoxicity in the stationary phase. MTS assay was performed and the percentage of cell viability was determined. 2.4 Cell uptake assay of plain and Mn-GNPs DU-145 cells (10 ml) were seeded in a cell culture dish containing 2×106 cells and cultured overnight. Once the cells were 70 % confluent, they were treated with plain and mannose functionalized 20 and 200 nm GNPs (50 μl). After 3 h of incubation, the unbound GNPs in the cell culture treatments were removed by washing the cells with PBS buffer twice. The cells were trypsinized with Trypsin– EDTA and centrifuged. After removal of the supernatant, the cells were resuspended in PBS to a final volume of 5 ml. At this stage, the total number of cells was quantified with a hemocytometer. Five milliliters of 50 % nitric acid (HNO3) was added to each sample to lyse the cells. Inductively
Cellular toxicity of colloidal gold in DU-145 cells
coupled plasma mass spectrometry was performed to measure the gold mass in the various samples. The total number of GNPs was calculated via the gold mass. The total number of GNPs in the solution was divided by the number of cells to determine the number of GNPs taken up by the cells (Connor et al. 2005). 2.5 Cytotoxicity of functionalized GNPs to DU-145 cells and E. coli ORN178 The cytotoxicity of functionalized Mn-GNP of 200 nm particles was carried out by adding 200 μl of DU-145 cells (1.5×106 cell per well) into a 96-well plate. After incubation at 37 °C for 24 h, the cells were treated with 200 nm MnGNPs, 200 nm Mn-GNPs with bacteria EC-178 and 208. A total of volume 50 μl of 200 nm Mn-GNPs was added. A preincubated mix, 1 ml of the 200 nm Mn-GNPs and 1 ml of the microorganisms was incubated at 37 °C with shaking for 1 h. The solution was centrifuged and washed with sterile PBS and resuspended in sterile PBS before adding to the 96well plates. Different plates were set up for 6-, 12-, 24-, 48-, and 72-h time periods. The MTS assay was performed and the cell viability was determined. 2.6 Protein corona of commercial 20 and 200 nm GNPs We wanted to study the protein adsorption of the commercial 20 and 200 nm GNPs to fetal bovine serum (FBS) in DU-145 cell culture media and DMEM. The 20 and 200 nm GNPs were incubated with DMEM and cell culture medium for 1 h at 37 °C. The 20 nm GNPs samples were spinned down at 10,000×g for 10 min. The soups from FBS and DMEM samples were saved. The pellet containing the particles was washed thrice with nanopure water at 10,000×g. The particles were finally resuspended in nanopure water. The 200 nm GNPs were spinned down at 2,655×g for 10 min. The soups from FBS and DMEM samples were saved. The pellet containing the particles was washed thrice with nanopure water at 2,655×g. The particles were finally resuspended in nanopure water. Samples of pure nanoparticles of both 20 and 200 nm GNPs, DMEM and cell culture medium were also included to compare the binding of the FBS proteins to the nanoparticle surface. SDS-PAGE was performed for all the samples using a 4–20 % gel (Biorad, USA). The gel was stained by Coomassie blue stain. A Smart Protein standard (Genscript, USA) was used to identify and analyze the presence of FBS bound to the samples. The gel bands were cut up and image taken accordingly. The hydrodynamic size of the plain GNPs and proteinbound GNP samples were measured using dynamic light scattering (DLS) using Malvern Zetasizer.
15
2.7 Statistical analysis All experiments were carried out in triplicates with results expressed as mean±standard error. Statistically significant differences were calculated using the two-tailed unpaired t test or one-way analysis of variance with p values of ≤0.05,
ORIGINAL PAPER
Size-dependent cellular toxicity and uptake of commercial colloidal gold nanoparticles in DU-145 cells Pallavi Vedantam & George Huang & T. R. Jeremy Tzeng
Received: 24 January 2013 / Accepted: 20 February 2013 / Published online: 9 March 2013 # Springer-Verlag Wien 2013
Abstract Urinary tract infection (UTI) is a predominant condition in prostate cancer patients. Escherichia coli ORN178 (EC-178) is the uropathogen that causes recurrent infection by binding specifically to adhesins of prostate cancer cells (DU-145 cells). Gold nanoparticles (GNPs) have been used in biodiagnosis of pathogens. In this study, we have investigated the binding time of EC-178 to DU-145 cells, the cytotoxicity and uptake of plain and mannose functionalized and 20 and 200 nm GNPs (D-mannan (Mn)GNPs). We also investigated the protein corona of GNPs when incubated with fetal bovine serum to study the protein corona which decides the biological fate of the GNPs. It was seen that EC-178 binds and is inside the DU-145 cells by 3 h of incubation period. Plain 20 nm GNPs decrease the percentage of viable cells in 48 and 72 h in log and lag phase of DU-145 cells. It was also observed that the Mn-GNPs were taken up by the DU-145 cells significantly more than the plain GNPs. Protein corona was observed when GNPs were incubated with fetal bovine serum which was confirmed by dynamic light scattering measurements and SDS-PAGE gel. Keywords Gold nanoparticles . E. coli . DU-145 cells . Mannose . Urinary tract infection . Cytotoxicity . Biodiagnostic . Protein corona . Cell uptake
1 Introduction The estimated risk of prostate cancer is 21 % and the lifetime risk of death is 2–5 %. Even though it can be diagnosed early and therapy can be started immediately, P. Vedantam (*) : G. Huang : T. R. J. Tzeng Department of Biological Sciences, Clemson University, Clemson, SC 29634, USA e-mail: [email protected]
patients developing metastatic conditions die. Apart from the potent issue of cancer, recurrent urinary tract infection (UTI) is one of the most prevalent symptoms (Klein and Thompson 2012). Escherichia coli ORN178 are the most common cause of UTI in humans. Various urovirulence factors of E. coli ORN178 have been identified such as molecular biology of surface receptors of the urothelial cells of the urinary tract, the adhesin specificity and primarily and the type 1 fimbriae associated with the organism (Sokurenko et al. 1997). Detection, diagnosis, and treatment of UTI play an important role in prostate cancer. One of the novel tools currently vastly studied are nanoparticles. Nanoparticles continue to be used as carriers for localized drug diffusion to treat and detect infections and diseases like cancer (Chen et al. 2008). Owing to their nano size, it is easy for these particles to diffuse into the cells and effect desired responses in treatment of diseases. However, the size, type, and surface charges of the particles play a vital role. Of the different kind of nanoparticles, gold nanoparticles (GNPs) have been extensively studied in this regard. It has been shown that E. coli ORN178 binds specifically to D-mannose, which is an integral part of the glycoproteins that are a part of the adhesive domain on host cells (Sharon 2006). The fimbriae of the uropathogenic E. coli ORN178 bind to the urolapkins on the surface of urothelial cells of the human bladder. We have demonstrated D-mannose functionalized 200 nm GNPs bind specifically to E. coli ORN178 (Vedantam et al. 2012). Continuing in the same direction in this study, we attempt to see if GNPs can be used as specific biodiagnostic tool to detect and treat UTI in prostate cancer cells (DU-145). This study investigates the binding time of E. coli ORN178 and E. coli ORN208 to DU-145 cells. It has been shown that E. coli ORN178 binds specifically to D-mannose only and E. coli ORN208 serves as a negative control as it has type 1 pili that fail to bind to D-mannose. In order to study the cytotoxicity of plain and functionalized 20 and 200 nm GNPs to
14
P. Vedantam et al.
DU-145, growth curve of the prostate cancer cells was done. Cytotoxicity tests in log and lag phase were performed to study their biocompatibility in vitro. Cellular uptake of GNPs was estimated and protein corona of GNPs was studied (Table 1).
Soy Broth with ampicillin (50 μg/ml). The cultures were washed and resuspended in sterile PBS. A 100 μl (3×108 cells/ml) aliquot of EC-178 and 208 was added to two wells each in the chamber slide. The slide was incubated for 1, 2, and 3 h. At the three different time periods, wells were washed with PBS and images were taken by a fluorescent scope (Zeiss LSM-510).
2 Materials and methods 2.2 DU-145 cell growth curve The strains E. coli ORN178 and 208 were provided by Dr. Chu-Cheng Lin, Department of Zoology, National Taiwan Normal University and were transformed with plasmid pGREEN by electroporation (Sambrook and Russell 2001). Two different GNPs: 20 and 200 nm were purchased from Ted Pella Inc., USA. The concentration of 20 nm GNPs was 7 × 1011 particles/ml and 200 nm GNPs was 7× 108 particles/ml. The sugar D-mannan (Mn) was purchased from VWR (USA). The sugar was dissolved in 0.3 M sodium phosphate buffer. Surface functionalization of GNPs with the Mn was carried out by a modified multistep procedure (Aslan et al. 2004; Vedantam et al. 2012). All chemicals required for functionalizing GNPs were purchased from VWR, USA. Human prostate carcinoma cell line DU-145 was graciously given by Dr Arun Sreekumar, Baylor College of Medicine, Houston, TX, USA. Dulbecco’s modified Eagle’s medium (DMEM) was modified to contain Earles Balanced Salt Solution, non-essential amino acids, 2 mM L-glutamine, 1 mM sodium pyruvate, and 1,500 mg/L sodium bicarbonate. It was supplemented with fetal bovine serum to a final concentration of 10 %, 100 UI/ml penicillin G, and 100 μg/ml streptomycin in a humidified incubator with 5 % at 37 °C. All the media components were purchased from Promega, USA. 2.1 Binding of E. coli to DU-145 cells An eight-well chamber slide was used to perform the bacterial cell adhesion assay to DU-145 cells. A total of 0.5 ml of DU-145 cells (1.5×106cells/ml) was seeded in each well. The chamber slide was incubated at 37 °C overnight for attachment and fresh media was added. Fresh cultures of E. coli ORN178 (EC-178) and E. coli ORN208 (EC-208) were cultured overnight in Tryptic Table 1 Hydrodynamic size distribution of GNPs Sample
Size (nm)
Zeta potential (mV)
20 nm GNP Protein coated 20 nm GNP 200 nm GNP Protein coated 200 nm GNP
24±2.1 66.61±2.3 213.5±2.9 261.9±5.3
−54.4 −18.2 −49.3 −13.3
Cells were plated 96-well microtiter plates at initial densities of 1,000, 2,000, 4,000, and 8,000 cells per well. The cell culture medium was changed every 3 days. Cell growth was tested by the CellTiter 96® AQueous One Solution Cell Proliferation Assay (MTS) purchased from Promega, USA. It is a colorimetric method for determining the percentage of viable cells that are proliferating. Briefly, the MTS tetrazolium is bioreduced by the viable cells into a colored formazan product which is stable and can be measured at an absorbance of 490 nm. The amount of formazan produced is directly proportional to the number of living cells in the well. MTS assays were performed every day after seeding until day8. All the experiments were carried out in triplicates. 2.3 Cytotoxicity of GNPs to DU-145 cells The cytotoxicity of plain GNPs was tested by CellTiter 96® AQueous One Solution Cell Proliferation Assay (MTS) purchased from Promega, USA. Cytotoxicity was measured in both the logarithmic and stationary phase of cell growth. For cytotoxicity measurements in the logarithmic phase, each cell line was incubated for 72 h in 96-well plate before adding the GNPs. Fresh medium containing increasing three different concentrations of the GNPs (10, 50, and 100 μl) was added to each well and the cells were incubated for 6, 24, 48, and 72 h time periods. The same was done for testing cytotoxicity in the stationary phase. MTS assay was performed and the percentage of cell viability was determined. 2.4 Cell uptake assay of plain and Mn-GNPs DU-145 cells (10 ml) were seeded in a cell culture dish containing 2×106 cells and cultured overnight. Once the cells were 70 % confluent, they were treated with plain and mannose functionalized 20 and 200 nm GNPs (50 μl). After 3 h of incubation, the unbound GNPs in the cell culture treatments were removed by washing the cells with PBS buffer twice. The cells were trypsinized with Trypsin– EDTA and centrifuged. After removal of the supernatant, the cells were resuspended in PBS to a final volume of 5 ml. At this stage, the total number of cells was quantified with a hemocytometer. Five milliliters of 50 % nitric acid (HNO3) was added to each sample to lyse the cells. Inductively
Cellular toxicity of colloidal gold in DU-145 cells
coupled plasma mass spectrometry was performed to measure the gold mass in the various samples. The total number of GNPs was calculated via the gold mass. The total number of GNPs in the solution was divided by the number of cells to determine the number of GNPs taken up by the cells (Connor et al. 2005). 2.5 Cytotoxicity of functionalized GNPs to DU-145 cells and E. coli ORN178 The cytotoxicity of functionalized Mn-GNP of 200 nm particles was carried out by adding 200 μl of DU-145 cells (1.5×106 cell per well) into a 96-well plate. After incubation at 37 °C for 24 h, the cells were treated with 200 nm MnGNPs, 200 nm Mn-GNPs with bacteria EC-178 and 208. A total of volume 50 μl of 200 nm Mn-GNPs was added. A preincubated mix, 1 ml of the 200 nm Mn-GNPs and 1 ml of the microorganisms was incubated at 37 °C with shaking for 1 h. The solution was centrifuged and washed with sterile PBS and resuspended in sterile PBS before adding to the 96well plates. Different plates were set up for 6-, 12-, 24-, 48-, and 72-h time periods. The MTS assay was performed and the cell viability was determined. 2.6 Protein corona of commercial 20 and 200 nm GNPs We wanted to study the protein adsorption of the commercial 20 and 200 nm GNPs to fetal bovine serum (FBS) in DU-145 cell culture media and DMEM. The 20 and 200 nm GNPs were incubated with DMEM and cell culture medium for 1 h at 37 °C. The 20 nm GNPs samples were spinned down at 10,000×g for 10 min. The soups from FBS and DMEM samples were saved. The pellet containing the particles was washed thrice with nanopure water at 10,000×g. The particles were finally resuspended in nanopure water. The 200 nm GNPs were spinned down at 2,655×g for 10 min. The soups from FBS and DMEM samples were saved. The pellet containing the particles was washed thrice with nanopure water at 2,655×g. The particles were finally resuspended in nanopure water. Samples of pure nanoparticles of both 20 and 200 nm GNPs, DMEM and cell culture medium were also included to compare the binding of the FBS proteins to the nanoparticle surface. SDS-PAGE was performed for all the samples using a 4–20 % gel (Biorad, USA). The gel was stained by Coomassie blue stain. A Smart Protein standard (Genscript, USA) was used to identify and analyze the presence of FBS bound to the samples. The gel bands were cut up and image taken accordingly. The hydrodynamic size of the plain GNPs and proteinbound GNP samples were measured using dynamic light scattering (DLS) using Malvern Zetasizer.
15
2.7 Statistical analysis All experiments were carried out in triplicates with results expressed as mean±standard error. Statistically significant differences were calculated using the two-tailed unpaired t test or one-way analysis of variance with p values of ≤0.05,
